Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Glutathione peroxidase is a selenoenzyme composed of four identical subunits of 21,000 Da which catalyzes the reduction of H.sub.2 O.sub.2 and other hydroperoxides (1, 2). In many tissues, such as the lens of the eye, glutathione peroxidase is the major defense against hydroperoxides (3). Researchers have implicated hydrogen peroxide damage as a major cause in the formation of cataracts. Glutathione 1 has the formula H.sub.2 N.gamma.Glu-Cys-Gly-OH and is utilized as a cofactor, supplying the electrons for the reductive reaction: EQU ROOH+2GSH.fwdarw.ROH+H.sub.2 O+GSSG (eq 1).
The X-ray crystal structure of GSH peroxidase has been determined with 0.2-nm resolution. The results from such analyses, as well as biochemical data have been used to develop a reaction mechanism for the reaction (2). The mechanism in FIG. 1 shows the selenium atom going from a selenol (E-Se-H) to a selenenic acid (ESeOH). In the presence of high concentrations of peroxide, it can be further oxidized to a seleninic acid (E-SeOOH). Thus, FIG. 1 illustrates the twostep reduction of selenic acid utilizes 2 mol of GSH/mol of enzyme.
Earlier work, in this laboratory, directed toward the development of compounds having GSH peroxidase activity, met with only limited success (4). This work was based on the design of small molecules which would mimic the structure of the active site of the enzyme containing the essential selenocystine residue. The most active compound produced in this study, 2, proved to be only 0.033 as active as the Ebselen compound, 3, the most active compound previously known (5). ##STR1##
Recently, a report by Reich and Jasperse (6) described the oxidation-reduction chemistry of 4 (FIG. 2). The results of this study suggest that two mechanistic pathways, A and B, are possible for the catalysis of 4 of the reduction of hydroperoxides by thiols. The following observations were especially interesting from a mechanistic viewpoint:
(1) under acid catalysis, the selenamide, 4, equilibrates with seleninamide, 8, and diselenide, 7. These compounds are produced by disproportionation of the selenenamide, 4;
(2) oxidation of 6 with MCPBA first gave the diselenide, 7, and then the seleninamide 8;
(3) oxidation of 4 produced the seleninamide, 8, and the oxidation rate was faster than the oxidation rate of the diselenide;
(4) under weakly acidic conditions, treatment of 4 with thiols gave the selenosulfide 5 and disulfide; and
(5) neither the selenosulfide 5 nor diselenide 7 reacted with thiol under neutral conditions. However, upon addition of a strong amine base (DBU), both gave the selenolate and disulfide.
These results suggest some guidelines for the construction of molecules showing GSH activity:
(1) more easily available diselenides would function as effectively as the more difficult to construct cyclic compounds in the production of the catalytically active species (observations 1-4 above); and
(2) inclusion in the molecule of a strongly basic group proximal to the active selenium atom is desirable as it would be expected to catalyze the reaction of thiols with the intermediate diselenide and selenosulfide. Presumably, the base functions to provide a source of nucleophilic thiolate anion (observation 5 above).
With these guidelines in mind, we chose as compounds for study, tertiary amines of the type 3 (FIG. 3). The use of tertiary amines seemed preferable to that of primary or secondary amines since intermediate 10and 14 are not stable compounds and are thus activated toward nucleophilic attack by thiol at the selenium atom.